A graduate student explores the microbial mysteries of quorum sensing

The 27th of January, at the entrance of the vast Bay of Bengal ... about seven o’clock in the evening, the Nautilus ... was sailing in a sea of milk. ... Was it the effect of the lunar rays? No: for the moon ... was still lying hidden under the horizon. ... The whole sky, though lit by the sidereal rays, seemed black by contrast with the whiteness of the waters. “It is called a milk sea,” I explained. —20,000 Leagues Under the Sea, Jules Verne, 1870

Throughout the vast oceans, silent conversations are regularly taking place among the Earth’s most primordial life forms. The multitudes of single-celled bacteria that inhabit the oceans have evolved a way to communicate with each other, come together, and coordinate their behavior. Bacteria talk, and in our lab at Woods Hole Oceanographic Institution, we are trying to eavesdrop on their chatter.

The process bacteria use has been dubbed “quorum sensing.” You can think about it this way: In Congress or a corporate boardroom, you need to muster a minimum number of individuals—a quorum—to conduct business or accomplish something together; the same is true in the microscopic realms of the ocean.

One example is collective defense: A bacterium, alone and floating in the ocean, is an easy target for a marauding protist. But itinerant bacteria have evolved ways to aggregate into tightly packed, highly organized, and usually slimy communities called “biofilms,” which attach to hard surfaces. Like walled cities, these biofilms protect the individuals living within them from assaults.

The good, the bad, and the slimy

Biofilms are everywhere, and they have wide-reaching impacts. If you have ever felt the plaque on your teeth, slipped on a slimy rock at the beach, or been frustrated by ugly green slime on the hull of your boat, you have had a close encounter with a biofilm.

In the human body, bacteria that cause dental cavities, ear infections, and fatal lung diseases in cystic fibrosis patients, for example, all forge biofilms. These act as a fortress to protect the bacteria from the body’s immune response or antibiotic treatments while they build their ranks and prepare to attack their host.

Similarly, biofilms serve as a refuge for disease-causing bacteria in the ocean. Biofilms transported on ships, the shells of marine animals such as lobsters, or on microscopic copepods can help spread and transmit pathogens such as cholera.

Biofilms are detrimental in other ways. They foul and clog water pipes. They form the substrate on which nuisance organisms such as barnacles settle on ship hulls. The U.S. Navy spends more than $100 million every year on fuel to overcome biofilm-induced drag on their vessels. In fact, much of the information we have about the structure and function of biofilms has been discovered by scientists interested in controlling and eradicating them. These include ways to prevent biofilm-forming infectious bacteria from building their biofilm fortress in the first place, leaving them susceptible to more traditional antibiotic approaches.

But biofilms also play essential, positive roles in a variety of critical natural processes and provide innumerable crucial ecoogical services. They provide habitat for beneficial bacteria, algae, and higher organisms. Swarms of bacteria detoxify pollutants in lakes, rivers, and oceans. By decomposing and recycling organic material, they help keep nutrients circulating in the food chain like money in financial markets.

Via behaviors regulated by quorum sensing, bacteria play a significant role in determining what happens to carbon in the ocean: whether it sinks to the depths and is buried as detritus on the seafloor, or converted into the greenhouse gas carbon dioxide and released back to the atmosphere.

How bacteria talk the talk

Bacteria communicate via “chemical” conversations. The “words” they use are small molecules. One variety of chemical “words” particularly common in the marine environment are modified amino acids called acylated homoserine lactones or AHLs, for short.

The basic process of quorum sensing is pretty simple. Bacteria are constantly producing a few AHLs, which diffuse through bacterial cell membranes into the environment. If no other AHL-producing bacteria are out there, the AHLs will diffuse away and quickly degrade, and bacterial “silence” will prevail.

But if enough AHL-producing bacteria are in the vicinity, the concentration of AHLs outside the bacteria will eventually rise. That’s the chemical signal to each bacterium that they’ve got a lot of buddies in the area. In this way, the bacteria sense that they have sufficient density, that they have achieved a quorum.

The communal buildup of AHLs triggers the production of more AHLs by individual bacteria, which keeps the process going—something called an autoinduction response. The AHLs bind to protein receptors, which interact with bacterial DNA and help to turn on genes inside all the bacteria throughout the quorum. The genes activate behaviors that would not have been worthwhile for an individual bacterium but are now collectively advantageous: They produce enzymes, secrete toxins, or produce the biochemical building blocks that will fortify the biofilm.

Ready, set, glow

In some bacteria, quorum sensing triggers the production of enzymes that catalyze light-producing reactions. The bacteria appear to glow! Swarms of bioluminescent bacteria are the presumed source of the “milky seas” that many sailors have periodically observed over the centuries and that Jules Verne described in 20,000 Leagues Under the Sea.

In fact, the phenomenon of bioluminescence sparked the discovery of quorum sensing 40 years ago, when researchers were studying how Hawaiian bobtail squids glow. They found that a bacterium, Vibrio fischeri, aggregates in the squids’ light organs in a symbiotic relationship. In exchange for a protected, nutrient-rich environment to live in, the bacteria do something as a community that they wouldn’t do as individuals: They glow.

The benefit that the bacteria get from bioluminescencing is still under debate, but we do know what’s in it for the squid. It counter-illuminates and camouflages their shadows when they are active on moonlit nights, so they aren’t as easily detectable by predators.

Quorum sensing research at WHOI

As it turns out, quorum sensing is a ubiquitous process in the types of bacteria that dominate marine microbial communities. With my research advisor, Ben Van Mooy, I have been using novel analytical approaches to try and home in on the fleeting chemical “words” used by marine bacteria.

I sample biofilms from all manner of marine environments in hopes of catching these conversations in action. I scrape slime from rocks I find on Vineyard Sound beaches, from ship hulls in local waters, and from sediments in the Chesapeake Bay. I isolate bacteria from seawater in the Indian Ocean and from the backs of marine organisms.

I bring these natural samples back to the lab, where we extract any AHLs present in the samples into organic solvents, just as you might make a cup of tea by extracting tea leaves with hot water. We then subject the extract to liquid chromatography, a process that can tease apart all the different molecules in the extract.

We then use a mass spectrometer to bombard the molecules with high energy, which smashes them into smaller pieces (think about what happens if you drop a ceramic dish onto the floor). By examining the size of each of these pieces, we can put them back together like a puzzle and identify the exact chemicals that were in the original extract.

Quorum-sensing bacteria are very sensitive to AHLs and so the molecules are usually present in very low concentrations. In the lab, scientists can cultivate very large batches of bacteria and isolate very large quantities of AHLs. However, in the environment, bacterial populations are so much smaller and dispersed over much wider areas. Both of these factors make it much more challenging for us to obtain detectable quantities of natural AHL.

But this isn’t the only obstacle that has limited scientists’ ability to eavesdrop on natural bacterial communities. AHLs are very sensitive to the acidity or alkalinity of seawater, and they degrade quickly in the ocean. In our lab, we have been measuring how quickly; it turns out that, on average, each AHL molecule self-destructs in just a few hours. So we have to make our measurements soon after we collect our samples from the ocean.

‘It is called a milk sea’

Our pot of gold would be a large-scale bacterial quorum in the ocean. Indeed, a recent and tantalizing observation suggests that large-scale, conspicuous bacterial quorums do exist. In 2005, scientists at the Naval Research Laboratory, Monterey Bay Research Institute, and the National Geophysical Data Center reported a “milky sea” that had occurred a decade earlier. It covered an area of 5,946 square miles in the Indian Ocean.

The Indian Ocean bioluminescence had all the characteristics that it was produced by bacteria. A huge bloom of phytoplankton had blanketed the ocean surface. Hordes of bacteria in the area massed onto the phytoplankton like swimmers on a fleet of rafts. When they sensed they had achieved a quorum, they signaled each other to glow, collectively bright enough to be seen by satellite.

To produce that much light, more than a billion trillion (1022, or 1 with 22 zeroes after it) cells would have had to be present in that 5,946 square miles. That’s quite a large quorum.

Now it is just a matter of time before we catch the ocean’s most conspicuous bacterial quorum in action. We hold out hope that we may be at the right place at the right time during research cruises we have planned over the next two years.

Meanwhile, our lab continues to make progress in deciphering the chemical conversations of bacteria within less spectacular, though no less significant, biofilms on marine particles and phytoplankton. Ultimately, our work may reveal how decisions made in tiny bacterial boardrooms have impacts that are felt throughout the vast oceans and atmosphere.

Laura Hmelo has been supported by a National Science Foundation graduate fellowship and the J. Seward Johnson Fund. The research was funded by the Office of Naval Research. This article was written during a science writing course for graduate students at WHOI, supported by funds from The Henry L. and Grace Doherty Professor of Oceanography.

Images

A 'MILKY SEA'—Bioluminescent marine bacteria aggregated off the coast of Somalia in January 1995 and illuminated a patch of ocean that was a bit larger than the state of Connecticut.
(Steven D. Miller, Naval Research Laboratory (NRL) and Steven H. D. Haddock, Monterey Bay Aquarium Research Institute; Christopher D. Elvidge, National Geophysical Data Center; and Thomas F. Lee, NRL.)

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